Composition for treating atopic dermatitis comprising extracts of bamboo and scutellaria

ABSTRACT

The present invention relates to a composition comprising of plant extract as an active component, specifically, Bamboo extract and  Scutellaria  extract, for the treatment and prevention of atopic dermatitis. 
     The present invention is a natural ingredient obtained from a plant. The present invention can control immune responses by inhibiting the release of histamine and leukotrien, and thus, has effect in the treatment or prevention of allergic diseases, inflammatory diseases and skin diseases, specifically atopic dermatitis. 
     The present invention has been proven safe and beneficial effecting the treatment of atopic dermatitis through clinical trials, and thus, can be used for the treatment and prevention of atopic dermatitis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/445,159, filed Apr. 28, 2009 (now U.S. Pat. No. 8,247,007); which isa U.S. National Phase Application of International Application No.PCT/KR2007/005004, filed Oct. 12, 2007; which claims priority to KoreaApplication No. 10-2006-0099182, filed Oct. 12, 2006. These applicationsare incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention is a composition comprising of a plant extract asan active ingredient for treating atopic dermatitis, specifically amixture composition comprising of Bamboo extracts and Scutellariaextracts.

BACKGROUND ART

Atopic dermatitis is an allergic disease caused by a defect of a stratumcorneum which is a protective wall located in the outermost part of theskin which is caused by hereditary, environmental, or immunologicalfactors and is exacerbated in arid climates. Many people are afflictedby the atopic dermatitis, specifically 0.5-1% of the total population.In cases of minors, 5-10% of children are afflicted by the atopicdermatitis. 50% of patients can recover by their second birthday, and25% can recover by puberty. However, 25% never recover and continue tosuffer from atopic dermatitis into adulthood.

The main symptoms of atopic dermatitis are severe pruritus, xeroderma,eruption or oozing of the skin, boils, scale like skin (scaly skin),etc.

The pathogenesis of atopic dermatitis is not completely understood, butgenetic factors are attributed to most cases of atopic dermatitis, andthe pathogenesis is related to immune response. It has been shown thatatopic dermatitis can be caused by a combination of dry skin, skin thatis prone to itching more than the average person, infections caused bybacteria virus fungi, etc., and emotional and environmental factors.

Specifically, an antibody (IgE) produced by a mast cell during thebody's process of naturally eliminating a material which causes a rashto form when in contact or invading the body causes a hypersensitivereaction when this same material invades the body again producing ahistamine which causes the atopic dermatitis. The mast cell isdistributed widely throughout organs such as, the skin, respiratoryorgans, mucosa of the gastrointestinal tract, circum of lymphatic duct,brain, and is known as the cell that causes diverse inflammation andallergic reactions. The histamine released from the mast cell causesinflammation and immediate allergic reaction by inducing vasodilation,smooth muscle-contraction of the gastrointestinal and/or bronchialtract, secretion of glandular cells, exacerbation of the reactions,etc., and serves as an intermediary for diverse biological effects suchas secretion of mucus and local protein.

Pharmacotherapies, such as steroids, anti-histamines, antibiotics areusually prescribed for atopic dermatitis. The steroid agent (adrenalcortical hormone agent) can act as an anti-inflammatory andimmuno-suppressant and has positive effect in treating the disease, butif used over a long period of time, side effects such as skin-weakening,symptom of systemic hormone, toxicity can result. Currently, uses ofimmune-suppression agents and novel anti-histamine agents have beenstudied for treating atopic dermatitis. However, anti-histamine agentscannot completely suppress the allergic reaction since other chemicaltransmitters in addition to the histamine can induce the allergicreaction. The mast cell releases other chemical transmitters such asleukotriene C4 and leukotriene B4 in addition to the histamine.Leukotriene C4 contracts the smooth muscle of bronchus like thehistamine, and leukotriene B4 causes chronic inflammation by inducingneutrophil and eosinophil and injures neighboring cells.

Thus, a novel composition for the effective treatment of atopicdermatitis without the side-effects is required.

Bamboo belongs to the Poaceae family. There are about 280 known speciesof bamboo all over the world, and about 70 species grow naturally or arecultivated in Korea. There are 11 representative kinds of Bamboo;Phyllostachys nigra, Phyllostachys bambusoides (Cedrela sinesis),Phyllostachys edulis (Phyllostachys puhescen), Phyllostachys nigra for.Punctata, Sasa borealis var. gracilis, Arundinaria simonii, Sasaborealis var. chiisanensis, Sasa borealis, Sasa albo-marginata,Pseudosasa japonica, etc. Among them, Phyllostachys bainbusoides(Cedrela sinesis), Phyllostachys nigra and Phyllostachys edulis arecultivated. According to Dongeui-Bogam, Compendium of Materia Medica andthe divine Farmer's Materia Medica, Bamboo is effective in treatingpalsy and hypertension, and was used to treat pneumonia and bronchitisto bring down fever, loosen phlegm and as a coolant. Recently, it hasbeen reported that Bamboo has been used to treat hypertension,atherosclerosis and cardiovascular disease. Bamboo is also known to haveanti-oxidant effect which is effective in the prevention of cancer andaging. Also, phytochemicals such as organic acid, dietary fiber, tannin,benzofuran within the plant are expected to contribute to preventingdiseases of the circulatory system.

The conventional studies for bioactive compounds focusing onantimicrobial activity have been reported mostly in Korean and Japan.Japanese researchers discovered the 2,6-dimethylbenzoquinone and benzoicacid which are antimicrobial compounds in the leaf of Bamboo, and KoreanPatent No. 10-0465113 discloses the effects of bamboo extract inimproving blood circulation and preventing inflammation. Japanese PatentPublication H09-278662 discloses fats and oils which have anti-allergiceffect contains the Bamboo extract obtained by using the soxhlet methodusing ether as a solvent, and WO 2002/07745 discloses that Bambooextract obtained by using water has antipruritic effect which iseffective in the treatment of atopic dermatitis.

Scutellaria has bioactive and pharmacological properties and has beenused in oriental medicine for treating fevers and allergies. It acts bydilating blood vessels and brings down blood pressure, and inhibitsatherosclerosis. Bicalin contained in Scutellaria is a kind of flavonoidwhich is effective to sedate or stop bleeding by suppressing thepermeability of capillaries. Also, bicalin inhibits the release ofchemical transmitters by strengthening the mast cell membrane and so cando anti-allergic action. Specifically, it is known that thepharmacological properties of Scutellaria are improving infectionscaused by allergies, inhibiting increased vascular permeability andalleviating inflammatory discharge of blood and congestion by stronganti-inflammatory effect, and these pharmacological properties arederived from bicalin. Bicalin is hydrolyzed to baicalein and glucuronicacid. Baicalein acts as a diuretic and glucuronic acid acts asdeintoxicant. Korean Patent Publication No. 1996-0003725 discloses atherapeutic agent comprising of the flavonoid ingredient of Scutellaria.Korean Patent Publication No. 1996-0040370 discloses a composition forthe prevention and treatment of alcohol disorder comprising ofScutellaria extract and flavone glycoside. Korean Patent Publication No.2002-0031608 discloses a Scutellaria extract that has positiveantimicrobial effect, and the process for preparing the extract and thepharmaceutical composition of the extract. Korean Patent No. 10-0522579discloses a mixture extract of Scutellaria and Omija (Schizandrachinensia Baillon) which has anti-stress effect.

The above properties of Bamboo or Scutellaria have been known, but therehas not been reported any therapeutic effect for atopic dermatitis usingthe mixture composition comprising of Bamboo extract and Scutellariaextract.

The inventors of the present invention have studied a novel compound forthe treatment of atopic dermatitis. As a result, they discovered andconfirmed that the mixture composition comprising Bamboo extract andScutellaria extract can strongly inhibit the release of histamine andleukotrien without any side-effects and has positive therapeutic effecton atopic dermatitis, to complete the present invention.

DISCLOSURE OF THE INVENTION

The objection of the present invention is to provide a compositioncomprising of a plant extract as an active ingredient which will have apositive therapeutic effect for the treatment and prevention of atopicdermatitis without any side-effects.

Also, the objection of the present invention is to provide a use ofmixture composition of Bamboo extract and Scutellaria extract for themanufacture of a medicament for the treatment and prevention of atopicdermatitis.

Also, the objection of the present invention is to provide a method ofthe treatment and prevention of atopic dermatitis by administering tothe subject a therapeutically effective amount of mixture composition ofBamboo extract and Scutellaria extract.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the improvement rate of clinical trial itemson antecubital space and popliteal space after administering the presentextract.

FIG. 2 is a digital steel photo showing the improvement effect of atopicdermatitis according to administration of the present extract bycomparing photos taken before and after using the product.

FIG. 3 is a graph showing measurement result of the moisture loss rate(g/m²h) which occurred per unit area and per unit time by usingTewameter TM300 (Courage+Khazaka, Germany) on 10 cm lower part ofpopliteal fossa and antecubital fossa at the time before the product wasused and after the product was used.

DETAILED DESCRIPTION OF THE INVENTION

To achieve the above objectives, the present invention provides acomposition for the treatment of atopic dermatitis comprising of Bambooextract and Scutellaria extract as an active ingredient.

Also, the present invention provides a use of mixture composition ofBamboo extract and Scutellaria extract for the manufacture of amedicament for the treatment and prevention of atopic dermatitis.

Also, the present invention provides a method of the treatment andprevention of atopic dermatitis by administering to the subject atherapeutically effective amount of mixture composition of Bambooextract and Scutellaria extract.

In the composition of the present invention, Bamboo is selected fromPhyllostachys, Sasa or Pseudosasa, and Phyllostachys is preferablyselected from the group consisting of Phyllostachys edulis,Phyllostachys nigra var. henonis, P. nigra, P. bainbusoides, P.pubescence, P. nigra for. punctata and P. comprossa, and Sasa ispreferably selected from the group consisting of Sasa coreana Nakai, S.coreana, S. kurilensis, S. quelpaertensis, S. borealis, S. borealis var.chiisanensis and S. borealis var. gracilis, and Pseudosasa is preferablyselected from Pseudosasa japonica and Pseudosasa japonica var.purpurascens.

In the composition of the present invention, for Bamboo and Scutellariacommercially purchased herbs can be used. The whole herb, branch, shell,leaf, sprout, root, endodermis, etc., can be used, preferably in theform of powder or extract.

The Bamboo extract and Scutellaria extract of the present invention canbe used by extracting Bamboo and Scutellaria with water, organicsolvent, or mixing solvents thereof. Although all conventional solventscan be used as the above organic solvent, polar solvent such as water,C₁₋₄ alcohol (such as methanol, ethanol etc.), etc., or mixing solventthereof is preferred. Preferably, water-insoluble fraction of 50-90% ofethanol extract or ethanol-soluble fraction of hot water extract can beused as the above bamboo extract.

The above extraction may be carried out by conventional methods such ashot water extraction, sonication, etc., and a lyophilized product of theextract can be used for the present composition. In addition, theextract can be further purified by conventional fractionation method orchromatography, and such fractionated material or purified material isalso within the scope of the present invention.

In the composition of the present invention, Bamboo or Scutellaria canbe used alone, but it is preferable to use a combined composition thatBamboo extract is additionally mixed with Scutellaria extract to showsynergistic effect.

In the composition of the present invention, the synergistic effect atthe time of administering the combination in comparison withadministration of the extract alone was measured and confirmed by usingthe COLBY formula (COLBY S. R., Calculating synergistic and antagonisticresponse of herbicide combinations, Weeds 15, 20-22, 1967).

As shown above, when the composition is used in combination with Bambooextract and Scutellaria extract, their weight ratios ofBamboo:Scutellaria could be in 1˜10:1˜10, but preferably 1˜5:1˜5, ormore preferably 1˜3:1˜3.

The composition of the present invention can be prepared intoconventional pharmaceutical preparations according to conventionalmethods in the pharmaceutical field, for example, solution such asdrinks, syrup, capsule, granule, tablet, powder, pill, ointment, andemulsion, skin external preparation such as gel, etc., by mixing it witha pharmaceutically acceptable carrier, excipient, etc.; and can beadministered orally or parenterally.

The composition of the present invention is appropriately administereddepending on the extent of absorption of the active ingredients into thebody; excretion rate; age, weight, sex, and condition of patient;severity of treated disease, etc. However, generally, the dosage for anadult is in solution 0.0001˜100 mg/kg, or preferably 0.001˜100 mg/kg,per day. It can be administered once a day or several times a day. Theamount should not limit the scope of the present invention in anymanner.

Hereinafter, the present invention will be described in more detail withreference to the following examples, but the scope of the presentinvention should not be construed to be limited thereby in any manner.

EXAMPLES Example 1 Preparation of Bamboo Extract Example 1-1 Preparationof Bamboo Ethanol Extract

Dried bamboo (20 kg) was extracted by adding 25% of ethanol (200 l) andheating the mixture at 80° C. for 6 hr. The extract was filtered andconcentrated to remove the ethanol until the extract volume reached 5 l.The concentrated extract was then cooled to room temperature. Thepellets were collected and dried to obtain the bamboo extract (390 g).

Example 1-2 Preparation of Bamboo Hot Water Extract

Dried bamboo (20 kg) was extracted by adding water in the amountequivalent to 10 times the weight of the dried bamboo and heating themixture at 100° C. for 4 hr. The extract was filtered and concentratedunder reduced pressure. The concentrated extract was added to ethanol(10 l) and stirred at 70° C. for 2 hr, and then cooled to roomtemperature. The pellets were filtered and concentrated under reducedpressure to obtain the bamboo extract (350 g).

Example 2 Preparation of Scutellaria Extract

Scutellaria (1 Kg) was added to water (8 l) and extracted by refluxingat 80° C. for 2 hr. The extract was cooled, filtered and concentrated,to obtain the Scutellaria extract powder (330 g).

Example 3 Preparation of Mixture Composition

The mixture composition was prepared by mixing Bamboo extract obtainedfrom Example 1 and Scutellaria extract obtained from Example 2. Theweight proportion of the Bamboo extract to the Scutellaria extractshould be 1:1, 1:2, 1:3 or 2:1, 3:1.

EXPERIMENTS Experiment 1 Measurement of Inhibition Activity of ReleasingHistamine and Leukotrien from the Mast Cell According to the Examples

The release of histamine and leukotrien from the mast cell is one of themajor causes for the allergic reaction. The effect of the mixturecomposition of the Bamboo extract and the Scutellaria extract ininhibiting the release of histamine and leukotrien from the mast cellwas measured.

Experiment 1-1 Isolation of the Mast Cell from Liver

Lung tissue (3 g/1 pig) was isolated from eight female guinea pigs (200g) and fat tissue, bronchus and blood were removed from the lung tissue.The isolated lung tissue was treated with enzyme (5 mg/ml ofcollagenase, 1.8 unit/27 μl of elastase) by using Tyrode TGCM buffercontaining Ca²⁺, Mg²⁺ and 0.1% of gelatin at 3 times for 15, 15, 25mins. The each enzyme treated lung tissue was filtered by nylon mesh andmetal mesh (100 μm), and then centrifuged (called ‘monodispersed mastcell’). The pellets was suspended with TG buffer (16 ml) containing 0.1%of gelatin, but no Ca²⁺ and Mg²⁺, and centrifuged by loading to roughPercoll (1.041 mg/ml density) at 1,400 rpm for 25 mins, to obtain thepellets. The pellets were re-suspended with TG buffer (8 ml) andcentrifuged by loading to discontinuous Percoll (1.06-1.10 mg/mldensity) at 1,400 rpm for 25 mins, to isolate several cell layers. Amongthe several cell layers, the third and fourth layers were washed twicewith TGCM buffer since the mast cell exists in third and fourth layers.The whole cell and mast cell were stained with trypan blue and alcianblue. The purity of the mast cell was measured by calculating the numberof cells, to obtain about 80-90% of the mast cell.

Experiment 1-2 Inhibition of Releasing Histamine from the Mast Cell

The mast cell (4105 cells) was treated with guinea pig IgG1 antibody(anti-OVA 1 ml/106 cells) at 37° C. for 45 mins, and washed with TGCMbuffer to remove anti-OVA antibodies which are not bound to the membraneof the mast cell. The mast cell was suspended with TGCM buffer (1 ml)and pre-treated with each reagents (30 μg concentration). The mast cellwas reacted by sensitizing using ovalbumin (1.0 μg/μl) for 10 mins,cooled in ice, and centrifuged, to measure histamine from the supernant.

The amount of histamine in each sample was measured by modifying themethod of Siraganian and using automated continuous-flow extraction anda flourometic analyzer (Astoria analyzer series 300, Astoria-pacificinternational, Oregon, USA). 1N-hydrochloric acid, 0.73M phosphoricacid, 5N sodium hydroxide, 1N sodium hydroxide, saline diluents andsampler wash, o-phthaladehyde solution was prepared and connected to atube linked to the analyzer. The storage solution of histamine wasdiluted to 20 ng, 10 ng, 5 ng, 3 ng and 1 ng, and theconcentration-dependent result of standard curve was obtained. Then,each sample was diluted with 2% of perchloric acid and the amount ofhistamine was measured. The result showed that the Bamboo extract andthe Scutellaria extract showed inhibition activity, respectively, andthe mixture composition of the Bamboo extract and the Scutellariaextract also showed high inhibition activity. The synergistic effect atthe time of administering the combination in comparison withadministration of the extract alone was measured and confirmed by usingthe COLBY formula (COLBY S. R., Calculating synergistic and antagonisticresponse of herbicide combinations, Weeds 15, 20-22, 1967) (Table 1).

TABLE 1 The inhibition activity of releasing histamine from the mastcell per each extract. Sample Inhibition activity (%) Control 32.5 ±0.25 Bamboo extract 22.4 ± 0.09 (31.1%) Scutellaria extract 26.4 ± 0.11(18.8%) Mixture composition (Bamboo:Scutellaria = 1:1) 10.1 ± 0.25(70.5%) Mixture composition (Bamboo:Scutellaria = 1:2) 15.4 ± 0.46(52.6%) Mixture composition (Bamboo:Scutellaria = 1:3) 19.7 ± 0.52(39.4%) Mixture composition (Bamboo:Scutellaria = 2:1)  9.3 ± 0.32(71.3%) Mixture composition (Bamboo:Scutellaria = 3:1) 13.2 ± 0.11(59.4%)

Experiment 1-3 Inhibition of Releasing Leukotrien from the Mast Cell

The amount of leukotrien in each sample was measured by using the methodof Aharoney et al. (Biochem. Biophys. Res. Commun., p 574-579, 1983).The leukotrien antibody was suspended with 5 mM MES buffer containing0.1% of gelatin, and to each tube the supernant of the cell (100 μl)which was treated with a reagent (30 μg) was added. The leukotrienantibody and [³H] leukotrien D4 (LTD₄) were added to the supernant andwas allowed to react at 4° C. for 2 hr. The reaction was stopped byusing dextran coated charcoal and the inhibition activity was measuredby using liquid scintillation spectrometry. The results showed that theBamboo extract and the Scutellaria extract showed inhibition activity,respectively, and the mixture composition of the Bamboo extract and theScutellaria extract also showed high inhibition activity. Thesynergistic effect at the time of administering the combination incomparison with administration of the extract alone was measured andconfirmed by using the COLBY formula (COLBY S. R., Calculatingsynergistic and antagonistic response of herbicide combinations, Weeds15, 20-22, 1967) (Table 2).

TABLE 2 The inhibition activity of releasing leukotrien from the mastcell per each extract. Sample Inhibition activity (%) Control 679.0 ±54.19 Bamboo extract 449.0 ± 40.47 (33.8%) Scutellaria extract 569.4 ±32.89 (16.1%) Mixture composition (Bamboo:Scutellaria = 1:1)  149.5 ±8.26 (78.0%) Mixture composition (Bamboo:Scutellaria = 1:2) 282.1 ±47.55 (58.5%) Mixture composition (Bamboo:Scutellaria = 1:3)  350.1 ±33.1 (48.4%) Mixture composition (Bamboo:Scutellaria = 2:1) 147.5 ±11.92 (78.3%) Mixture composition (Bamboo:Scutellaria = 3:1) 322.9 ±33.65 (52.4%)

Experiment 2 Clinical Trials

20 patients suffering from severe atopic dermatitis were tested by usingthe mixture composition of Bamboo extract and Scutellaria extractselected from Experiment 1 for 4 weeks. The present composition wasspread onto the popliteal fossa and the antecubital fossa, and theresults were investigated.

In the clinical trial, the effects before and after using the productwere estimated by using the Local SCORAD index. The results wereestimated by rating the degrees of 6 intensity items, erythema,edem/population, oozing/crusting, excoriation, lichenification, drynesson a scale of 4 (0=absent, 1=mild, 2=moderate, 3=severe) for the rightand left side of popliteal fossa and antecubital fossa which were thenused to show improvement rate.

The results showed that there was improvement effect after using theproduct, specifically there was more than 50% improvement in erythema,oozing/crusting and excoriation (FIG. 1). The result was photographed byusing a Digital Still Camera (DSC-S75, Sony) at the time before theproduct was used and after the product was used (FIG. 2). Also, themoisture loss (g/m²·h) due to evaporation which occurred per unit areaand per unit time was estimated by using Tewameter TM300(Courage+Khazaka, Germany) on 10 cm lower part of popliteal fossa andantecubital fossa at the time before the product was used and after theproduct was used. The moisture loss on trandermal was reduced each time,specifically the improvement on the antecubital fossa was better than onthe popliteal fossa (FIG. 3).

INDUSTRIAL APPLICABILITY

The present invention is a natural ingredient obtained from a plant, andcan control immune responses by inhibiting the release of histamine andleukotrien. It has been confirmed that the present invention is safe andis beneficial to the treatment of atopic dermatitis, and thus, thecomposition can be used for the treatment and prevention of atopicdermatitis.

1.-16. (canceled)
 17. A method for treating allergic skin disease,comprising administering to a subject a therapeutically effective amountof a composition comprising a Bamboo extract and a Scutellaria extract,wherein the weight ratio of Bamboo extract to Scutellaria extract rangesfrom 1:3 to 3:1.
 18. The method according to claim 17, wherein theBamboo extract is a water-insoluble fraction of a 50-90% ethanolextract.
 19. The method according to claim 18, wherein the Scutellariaextract is obtained by extraction with water, methanol, ethanol or amixture thereof.
 20. The method according to claim 17, wherein theBamboo extract is an ethanol-soluble fraction of a hot water extract.21. The method according to claim 20, wherein the Scutellaria extract isobtained by extraction with water, methanol, ethanol or a mixturethereof.
 22. The method according to claim 17, wherein the compositioninhibits a release of histamine, leukotriene, or a combination thereof.23. The method according to claim 17, wherein the method comprisestreating atopic dermatitis.
 24. The method according to claim 17,wherein the method comprises treating erythema, edema, papulation,oozing, crust, excoriation, lichenification or dryness.
 25. The methodaccording to claim 17, wherein the method decreases transdermal moistureloss.
 26. The method according to claim 17, wherein the composition isformulated with a pharmaceutically acceptable carrier or excipient. 27.The method according to claim 17, wherein the composition isadministered topically.
 28. The method according to claim 17, whereinthe composition is prepared as a gel, ointment, or emulsion.
 29. Themethod according to claim 28, wherein the composition is administeredtopically.